RESEARCH ARTICLE Open Access

Molecular phylogeny of the Palaearcticbutterfly genus Pseudophilotes(Lepidoptera: Lycaenidae) with focus onthe Sardinian endemic P. barbagiaeValentina Todisco1, Andrea Grill1, Konrad Fiedler1, Brigitte Gottsberger1, Vlad Dincă2,3, Raluca Vodă4,Vladimir Lukhtanov5,6 and Harald Letsch1*

Abstract

Background: The Palaearctic butterfly genus Pseudophilotes Beuret, 1958 (Lycaenidae: Polyommatinae), that todayoccurs in North Africa and in Eurasia, includes ten described species with various distribution ranges, includingendemics such as the Sardinian P. barbagiae. Phylogenetic relationships among these species are largelyunresolved. In the present study, we analysed 158 specimens, representing seven species out of ten described inthe genus, from widely distributed sites throughout the western Palaearctic region, using nuclear markers (28SrRNA, wingless, Internal Transcribed Spacer 2 and Elongation Factor 1α) and the barcoding region of themitochondrial cytochrome c oxidase subunit 1 gene to investigate if the current taxonomic entities match thephylogenetic pattern. Further, we attempt to infer the geographic origin of the genus Pseudophilotes and estimatethe timing of its radiations, including the split of the Sardinian endemic P. barbagiae.

Results: Maximum Likelihood and Bayesian inference analyses confirmed the monophyly of the genusPseudophilotes and clearly supported the closer affinity of P. barbagiae to the species assemblage of P. baton, P.vicrama and P. panoptes as opposed to P. abencerragus. The currently accepted species P. baton, P. vicrama and P.panoptes turned out to be weakly differentiated from each other, while P. bavius and P. fatma emerged as highlydistinct and formed a well supported clade. The split between the lineage comprising bavius and fatma (sometimestreated as a distinct genus, Rubrapterus) with Salvia species as larval host plants, and the remaining Pseudophilotesthat utilize Thymus and other Lamiaceae (but not Salvia), dates back to about 4.9 million years ago (Mya).

Conclusions: Our results show that the last common ancestor of the genus probably lived in the Messinian period(5.33–7.25 Mya). At species level, they support the current taxonomy of the genus, although P. panoptes, P. batonand P. vicrama display complex patterns based on phylogeographic relationships inferred from mtDNA. TheSardinian endemic P. barbagiae turned out to be a young endemic, but clearly with European instead of NorthAfrican origin and evolved through allopatric isolation on the island of Sardinia only about 0.74 Mya.

Keywords: Pseudophilotes, Lycaenidae, Butterfly, Sardinia, Endemism, Messinian Salinity Crisis, Pleistocene, Pliocene

* Correspondence: harald.letsch@univie.ac.at1Department of Botany and Biodiversity Research, Division of TropicalEcology and Animal Biodiversity, University of Vienna, Rennweg 14, A-1030Vienna, AustriaFull list of author information is available at the end of the article

BMC Zoology

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Todisco et al. BMC Zoology (2018) 3:4 https://doi.org/10.1186/s40850-018-0032-7

BackgroundPseudophilotes Beuret, 1958 (Lycaenidae: Polyommatinae)is a Palaearctic genus of blue butterflies mainly distributedin Eurasia, but also in North Africa and the Levante(Fig. 1a). The phylogenetic position of the genus withinthe Glaucopsyche section (Polyommatinae) has been lately

inferred by Ugelvig et al. [1]. They found that the generaPseudophilotes and Scolitantides are clearly separated,although they had been lumped together by other studies[2, 3]. This separation is explained by the affiliation withdifferent larval host plants: Pseudophilotes with Lamia-ceae, and Scolitantides with Crassulaceae.

a

b

Fig. 1 a Sampling localities and approximate geographic distribution areas of the Pseudophilotes species considered in the present study.Sampling localities for the species included in this study are showed with differently coloured circles, diamonds, and triangles respectively.Geographic distribution areas are redrawn after [2, 3, 26, 74–77] and shaded in colours referring to the sample localities. The map was preparedusing Quantum GIS 2.8.2 based on a map from Natural Earth (www.naturalearthdata.com). b Median-Joining Network of Pseudophilotes COIsequences. The circle size is proportional to haplotype frequency and the different colours refer to the different species used in the study (cf. a).Numbers of mutations between haplotypes are shown at the connections, except for single or double substitutions. Connections creating loopsthat are less probable according to frequency and geographical criteria are indicated in dashed lines. Haplotype codes match those inAdditional file 1. A1 - A3: haplogroups including P. baton, P. vicrama and P. panoptes

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At the species level, the same study [1] showed thatthe Bavius Blue, Pseudophilotes bavius forms a sisterclade to the rest of Pseudophilotes, which is also sup-ported by its association to Salvia (Lamiaceae), whereasspecies in the remaining clades mainly feed on Thymus(Lamiaceae). Recent efforts to resolve the evolution ofPseudophilotes based on the COI barcoding region [4]indicated a closer relationship of the Sardinian Blue, P.barbagiae to the False Baton Blue, P. abencerragus thanto the Baton Blue, P. baton. Further studies based onmtDNA [5] showed P. baton and the Eastern BatonBlue, P. vicrama to display complex patterns. Species de-limitation in the genus remains thus a work-in-progress.Species such as the Sardinian Blue, Pseudophilotes bar-

bagiae [6], with highly restricted distributions and taxarepresenting unique lineages within a species flock canbe suitable models to investigate the processes of differ-entiation and speciation and often provide the key foranswers to broader-scale biogeographic questions [7]. Inthis respect, the study of islands where many endemicspecies or subspecies evolved has provided fundamentalinsights into the relationships between geographicalpatterns and evolutionary processes [8, 9]. P. barbagiaehas been described as distinct from the continental andCorsican P. baton by characteristics of the male genitaliaand wing markings. Its divergence has been hypotheticallylinked to the geographic isolation of the Sardo-Corsicanplate [10], but this assumption has never been tested onthe basis of molecular data. Gerace [11] hypothesized thatP. barbagiae probably diverged from P. baton as a resultof the separation of Corsica and Sardinia due to importantclimatic changes, and later P. barbagiae became isolatedin mountainous areas.Due to its geographic position and geological history,

the island of Sardinia is characterized by a remarkablerichness of endemic species [12] and represents one ofthe most prominent biodiversity hotspots in the Medi-terranean basin [13]. The question how its endemicfauna was formed and when it originated has been previ-ously investigated using molecular analyses for a variety ofvertebrate [14–16] and invertebrate animals [9, 17–21]. Inall these cases, phylogenetic patterns could be related toseveral aspects of the geological history of the Mediterra-nean basin [11, 12], but the specific evolutionary historiesturned out to vary distinctly between taxa. About 5 Myaago, during the Messinian period, the closure of the Straitof Gibraltar led to an almost complete desiccation of theMediterranean Sea, and to a subsequent formation of saltdeposits in all deep basins [22]. This event, called theMessinian Salinity Crisis (MSC), was often advocated asone of the main drivers for the emergence of a distinctivefauna and flora in the Mediterranean. The newly exposedland corridors permitted faunal exchange between south-ern Europe and the Maghreb region (Northern Africa)

[23, 24], as well as between Sardinia and northern Italyand southern France.When the connection to the Atlantic ocean was

re-established, the massive influx of water brought abouta drastic change in marine flora and fauna, and at thesame time terminated the connections between the Tyr-rhenian islands and the Eurasian or African continentalmainland, respectively, which had as a consequence thedifferentiation of many distinctly younger island species(e.g. butterflies, cave-beetles, mayflies and lizards). Landbridges between Sardinia and Corsica and the mainlandcontinued to be created well into the Pleistocene due toglacial/interglacial sea-level oscillations [25]. During theWürm glaciation (115,000–11,700 years ago), Sardiniacould have been in contact with the mainland via theisland of Elba as the sea level was up to 120 m lowerthan today [11, 12] and with the neighbouring Corsica.In the present study, we analysed four nuclear genes

(28S ribosomal [28S], wingless [wg], Internal TranscribedSpacer 2 [ITS2] and Elongation Factor 1α [EF1α]) andthe barcoding region of the mitochondrial cytochrome coxidase subunit 1 (COI) gene for seven out of ten Pseu-dophilotes species, from widely distributed sites through-out the Palaearctic region, including for the first timethe Sardinian endemic P. barbagiae, as well as the Afri-can P. fatma. We aimed to (i) reconstruct the phylogenyof the genus Pseudophilotes and check if the obtainedclades correspond to current taxonomic units, (ii) totrace the geographical origin of the genus since extantdistributions of Pseudophilotes species cover both Eurasiaand North Africa, and (iii) to shed light on the historicalbiogeographic processes that determined the present-dayisolation of the Sardinian endemic P. barbagiae.

MethodsTaxon sampling and molecular proceduresWe examined 96 Pseudophilotes individuals representingseven species out of ten described from sites acrossEurope and North Africa (Maghreb region) (Fig. 1a andAdditional file 1). We were unable to obtain sequencesfrom P. sinaicus, P. jordanicus and P. jacuticus. Weincluded sequences from the Eurasian species Scolitan-tides orion Pallas, 1771 in our analysis, since the Pseudo-philotes species complex is frequently attributed torepresent just a clade within a more inclusive genusScolitantides Hübner, (1819) [2, 26].Specimens had either been dried or stored in 99%

ethanol after collection. Subsequently, samples werestored at − 20 °C until DNA extraction. The selectedmitochondrial (mt) and nuclear (nc) genetic markers(COI, 28S, wg, ITS2, EF1α) were amplified with varyingsuccess. When necessary, new specific PCR primerswere used (see Additional file 2), as the specimens hadbeen collected between the years 2000 and 2014; older

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samples performed often worse in Polymerase Chain Re-action (PCR) than the more recent ones.Extraction and amplification of mt and nc DNA was

conducted in two different laboratories, the CanadianCentre for DNA Barcoding (CCDB) (University ofGuelph, Guelph, Canada) and the Department of Botanyand Biodiversity Research (University of Vienna,Austria), according to the following procedures. In theCCDB lab, total DNA was extracted from one leg ofeach individual on Biomek FX liquid handling robotusing a semi-automated DNA extraction protocol [27]on glass fiber plates (PALL Acroprep 96 with 3 μm GFmembrane over 0.2 μm Bioinert membrane). DNA waseluted in 35-40 μl of ddH20 pre-warmed to 56 °C andstored at − 80 °C. PCR and sequencing for mtDNA werecarried out following standard procedures for Lepidop-tera [28, 29]. A fragment of 658 bp of the mt COI genecorresponding to the barcoding region was obtained. Inthe Vienna lab, total DNA was extracted from legs ofbutterflies homogenized with ceramic beads using aPrecellys 24 homogenizer set to 5000 min-1 for 2x20s.DNA extraction was performed according to the stand-ard protocol supplied with the Analytik Jena innuPREPDNA Micro Kit (Analytik Jena AG). Four nuclear genefragments were amplified using the Fermentas PCR sys-tem. PCR reactions were set up as follows: 2.5 μl of 10×(NH4)2SO4 PCR buffer, 2.5 μl MgCl2 (25 mM/l), 0.5 μldNTPs (10 mM/μl), 0.5 μl of each primer (10 pg/μl),0.2 μl BSA, 1 μl template DNA, 0.2 μl Taq polymeraseand filled to 25 μl with PCR-grade H2O. PCR reactionswere purified by digestion with shrimp alkaline phos-phatase and exonuclease for 15 min at 37 °C followed by15 min at 80 °C for enzyme deactivation. Sequencing re-actions were set up with 1 μl ABI BigDye 3.1 (AppliedBiosystems, Carlsbad, CA, USA), 1 μl primer, 1.5 μlsequencing buffer, 1 μl template DNA and filled to 10 μlwith PCR grade H2O. Primer names, references, primersequences as well as respective annealing temperatureand time are shown in Additional file 2. Sequences wereobtained by using an ABI capillary sequencer followingthe manufacturer’s recommendations. All gene frag-ments were sequenced in both directions and they wereedited and assembled using CodonCode Aligner 6.0.2.All sequences were submitted to GenBank (AccessionNumbers in Additional file 1) and in BOLD systemrepository (dataset name: DS-PSEUDOPH; DOI:dx.doi.org/10.5883/DS-PSEUDOPH).

Data set compilation and tree reconstruction proceduresTo reconstruct the phylogenetic relationships of Pseudo-philotes, the 96 specimens sequenced in our labs werecomplemented by 62 publicly available specimens ofPseudophilotes and 53 outgroup species of the subfamilyPolyommatinae and two species of the subfamily

Lycaeninae, mainly selected from Vila et al. [30](Additional file 1).All mt and nc protein coding genes were aligned using

CodonCode Aligner 6.0.2. The nc ribosomal 28S rRNAgene fragment and the nc ITS2 sequences were alignedwith the software MAFFT v7.245, using the iterativerefinement method G-INS-i [31] via the MAFFT onlineserver (http://mafft.cbrc.jp/alignment/server/). As the ncITS2 region consists of highly variable sections, its align-ment remained partly ambiguous. We therefore used thesoftware Aliscore v.2.0 [32] to identify ambiguouslyaligned or randomly similar sections (RSS) within the ncITS2 alignment. Aliscore was applied with the followingconditions: gaps were treated as ambiguous characters, awindow size of six positions was selected and themaximum number of pairs was compared to check forreplications. Taken together, the resulting alignments ofall mt and nc target fragments consisted of 3929 bp.Haplotype diversity and the number of variable sites of

mt and nc target fragments were calculated using DnaSP5.0 [33]. To represent the genealogical relationshipsamong mtDNA haplotypes we calculated a Median Join-ing (MJ) network using NETWORK 5 [34].Pseudophilotes relationships were assessed via Max-

imum Likelihood (ML), including all mt and nc targetfragments (3929 bp), and using the tree reconstructionmethod IQ-TREE v.1.61 [35]. Prior to analysis, the dataset was subdivided into eleven partitions, according tocodon positions of the protein coding genes and thesingle ribosomal genes. To reduce over-parameterizationand increase model fit, these partitions were furthermerged if possible and for each of the remaining parti-tions, the best fitting model was estimated withinIQ-TREE, which resembles the partitioning algorithm ofthe software PartitionFinder v2.0 [36]. Node support wasassessed by ultrafast bootstrap (UFBoot) analysis [37]with 1000 replications.

Multilocus coalescent species deliminationWe tested the status of all Pseudophilotes species ac-cording to current taxonomy with the multilocus Bayes-ian Phylogenetics and Phylogeography software (BPPv4.0; [38]). Within BPP we employed the joint speciesdelimitation and species-tree inference approach, forwhich the species tree inferred from the ML tree recon-struction was used as a guide tree. Prior to these ana-lyses, we excluded all outgroups except Glaucopsyche,Philotes and Scolitantides, which resulted in a condenseddata set, comprising 169 specimens and 3253 bp. BPPnow implements inverse gamma priors for the parame-ters theta (θ) and tau (τ). This allows integrating out θestimates analytically, which helps with moves betweentrees and delimitation models. We used an inversegamma prior with mean 0.002 for θ and 0.001 for τ. The

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analyses were run for 200,000 generations with aburn-in of 10,000 generations and a sample frequency offive and were run three times to confirm consistencybetween runs.

Divergence times estimationsDivergence times of the species were further assessedusing a species tree approach on the condensed data setin *BEAST 1.8.2. [39]. Each Pseudophilotes specimen inthe dataset was assigned to one of the potential speciesdelimited with a posterior probability of > 0.95 in BPP.The condensed dataset was further partitioned into thefive gene fragments, which were subsequently loadedinto PartitionFinder v2.0 to find the best fitting modelscheme for each gene. In *BEAST, all substitution, clockand tree models were unlinked. We assigned Brower’srate of 0.0115 substitutions/site/million years [40] to theCOI partition and estimated the rates of the other parti-tions. A lognormal relaxed clock with uncorrelated rateswas assigned to each clock model. The Tree model wasset to “Speciation: Yule Process”. *BEAST analyses wererun on the CIPRES Science Gateway v.3.3 [41], consist-ing of 150 million generations with sampling every10,000 generations. The first 75 million generations werediscarded as burn-in and convergence and mixing of theparameters were checked in Tracer 1.6 (http://tree.bio.e-d.ac.uk/software/tracer/). Post-burnin samples were sub-sequently used to construct a maximum clade credibilitytree in TreeAnnotator [42].

Tests of demographic equilibrium and populationexpansion in the mtDNA haplogroupTo test demographic equilibrium or events of recent ex-pansion in the genus, different sets of mtDNA sequenceswere selected (Table 1) on a geographical basis and tak-ing into account the results of the previous phylogeneticanalysis. Demographic equilibrium was tested by calcu-lating FS [43] and R2 [44] parameters, which have bothbeen shown to provide sensitive signals of historicalpopulation expansions [44]. Arlequin 3.0 [45] andDnaSP 5.0 [33] were used to compute FS (P < 0.02) and

R2 (P < 0.05) respectively, and to test their statisticalsignificance by simulating random samples (10,000 repli-cates) under the null hypothesis of selective neutralityand constant population size using coalescent algorithms(both modified from Hudson [46]). Expected mis-matched distributions and parameters of sudden expan-sion τ = 2 μ t (τ, sudden demographic expansionparameter; t, true time since population expansion; μ isthe substitution rate per gene) were calculated usingArlequin 3.0 by a generalized least-squares approach[47], under models of pure demographic expansion andspatial expansion [48, 49]. The probability of the dataunder the given model was assessed by the goodness-of-fittest implemented in Arlequin 3.0. Parameter confidencelimits were calculated in Arlequin 3.0 through a paramet-ric bootstrap (1000 simulated random samples).

Biogeographical analysesWe applied the R software package BioGeoBEARS v.0.21[50, 51] to infer the biogeographical history of Pseudo-philotes and to test the support for its origin either inEurasia or in North Africa. BioGeoBEARS uses theLagrange DEC model [52, 53], as well as likelihood in-terpretations of the DIVA model [54] and the BayAreamodel [55]. For each of these biogeographical models, itfurther implements a parameter describing founder-event speciation (+j). All model setups were comparedin a statistical framework allowing the selection of thebest-fitting model. The analyses were conducted withthe BEAST maximum clade credibility tree (MCC) fromthe *BEAST analyses, excluding the outgroup speciesGlaucopsyche spp. This was necessary, as the relation-ships within this genus are still ambiguous [1] and there-fore the unclear primary distribution of Glaucopsyche(Nearctic vs. Palaearctic) would have blurred the biogeo-graphical reconstruction of Pseudophilotes. The follow-ing areas were designated (see Fig. 2c): Eurasia (A),Iberian Peninsula (B), North Africa (C), Sardinia (D),and North America (E). The maximum number of an-cestral areas was set to three in both sets of analyses.

Table 1 Results of the tests of demographic equilibrium and mismatch analysis in mtDNA phylogeographic groups

Group N H h Fs P R2 P τ τ (5%) τ (95%) t (ka) t (5%) t (95%)

P. abencerragus + P. barbagiae 36 21 0.88 ± 0.04 −5.647 0.03 0.117 0 – – – – – –

P. abencerragus 23 19 0.98 ± 0.02 −11.694 0 0.127 0 – – – – – –

P. bavius 21 7 0.82 ± 0.063 −2.466 0.05 0.143 0 – – – – – –

A1 + A2 + A3 98 20 0.80 ± 0.027 −5.878 0.02 0.093 0 – – – – – –

A2 40 9 0.56 ± 0.087 −5.045 0 0.126 0 0.623 0 3.129 41 0 207

A3 21 9 0.85 ± 0.06 −2.852 0.04 0.138 0 – – – – – –

Significantly small values of FS and R2 are in boldN number of mtDNA sequences, H number of unique mtDNA haplotypes, h mtDNA haplotype diversity (±SD), FS Fu’s FS statistic, R2 Rozas and Ramon-Onsins’ R2statistic, τ sudden demographic expansion parameter, with 5 and 95% confidence limits, t true time since population expansion, from τ = 2 μ T (where μ is thesubstitution rate per gene)

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ResultsGenetic diversityFor COI (658 bp), 158 sequences (96 sequences analysedin this study plus 62 sequences retrieved from GenBank)showed 50 different haplotypes (Fig. 1b; Additional file 1)with 64 variable sites. The total haplotype diversity (h)was 0.917 (±0.013 Standard Deviation (SD)). The thirteensequences of P. barbagiae displayed two mtDNA haplo-types. P. abencerragus sequences displayed 19 mtDNA hap-lotypes (23 sequences) and h was 0.98 (±0.02 SD) (Table 1).The polyphyletic group including sequences from P. baton,P. vicrama and P. panoptes (haplogroups A1 - A3; Fig. 1b)

displayed 20 mtDNA haplotypes (98 sequences) and h was0.80 (±0.027 SD). The 37 28S sequences (832 bp) displayed17 haplotypes and 20 variable sites. The 31 wg sequences(403 bp) displayed 19 haplotypes and 34 variable sites. The35 EF1α sequences (1154 bp) displayed 72 variable sites.The 25 ITS2 sequences (627 bp) displayed 13 haplotypesand 36 variable sites.

Phylogenetic reconstruction and coalescent speciesdelimitationThe ML tree reconstruction (see Additional file 3) re-covered a clade consisting of Philotes sonorensis and

c

a b

Fig. 2 a Chronogram obtained from *BEAST species tree analyses of the COI, ITS2, wg, 28S and EF1α sequences. Nodal scale bars indicate the95% highest posterior density interval for divergence time estimates in Mya. Black nodes: pP ≥ 0.95; white node: pP < 0.95. b Ancestral areareconstruction results of the best BioGeoBEARS model (BayArea+j). The letters preceding the species indicate the distribution of extant species. cMap (from Natural Earth: www.naturalearthdata.com) showing four biogeographical regions in different colours, as defined in this study.Biogeographical region abbreviations: Eurasian (A), Iberian Peninsula (B), North Africa (C), and Sardinia (D). The four images on the right side ofthe figure show representatives of the genus Pseudophilotes. Photos: V. Dincă

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Scolitantides orion as the closest relative to Pseudophi-lotes (Bootstrap support, BS = 98). Our results furthersupport the monophyly of the genus Pseudophilotes (BS= 96) recognizing two well-supported main clades. Oneof them comprised P. bavius (distributed from the Bal-kan Peninsula and Romania eastwards to Turkey, south-ern Russia and Iran), and the Maghrebian endemicspecies P. fatma (BS = 100). In the other clade (BS =100), P. abencerragus (distributed from Israel across theMaghreb into the Iberian Peninsula) was recovered assister to all remaining species. This latter clade (BS = 96)was split into the Sardinian P. barbagiae (BS = 100) anda clade comprising P. vicrama, P. baton and P. panoptes(BS = 90). Although this clade shows three distinctgroups, all species accepted in current taxonomy ap-peared polyphyletic with some members of P. panoptesscattered in the P. baton clade and members of P. batonappearing in P. vicrama. In contrast, the species delimi-tation analyses in BPP recovered all different taxonom-ical units of Pseudophilotes as species with highestposterior probabilities (pP = 1.0).

Divergence time estimationsThe relationships inferred with ML were congruent withthe results of the *BEAST analyses (Fig. 2a), except forthe position of P. vicrama, which appeared as sistergroup to P. panoptes in the ML analyses (although withlow support, Additional file 3) and as sister group to P.baton in the *BEAST analyses (well supported, Fig. 2a).The chronogram in Fig. 2a shows the median ages and95% highest probability density (95% HPDs) computedfrom the post burn-in posterior topologies. We esti-mated the origin of the genus Pseudophilotes at 5.71Mya (95% HPD: 3.24–9.13 Mya). The split between P.bavius and P. fatma was estimated at 1.65 Mya (95%HPD: 0.79–3.13 Mya). The split between P. abencerragusand the remaining species was estimated at 1.25 Mya(95% HPD: 0.59–2.24 Mya) and finally the emergence ofP. barbagiae was estimated at 0.74 Mya (95% HPD:0.32–1.32 Mya).

Geographic distribution of mtDNA haplotypesThe mtDNA haplotype network (Fig. 1b) highlightedhigh haplotype diversity for P. abencerragus (0.98 ± 0.02)and P. panoptes (0.85 ± 0.06; haplogroup A3) and astar-like configuration of the haplogroup A2, where thecentral haplotype H32, including P. vicrama haplo-types distributed from Finland to Iran, was alsoshared with P. baton from Corsica and southern Italy(see Additional file 1). The H23 haplotype, includingalmost all P. baton sequences, is instead widely dis-tributed from Spain to southern Italy and was alsoshared with two P. panoptes individuals, similarly tothe H22 haplotype. Besides two haplotypes (H22 and

H23) shared with P. baton, the analyses highlightedthat P. panoptes (A3) owns nine characteristic (i.e.not shared with any other taxon) mtDNA haplotypes.The thirteen sequences of P. barbagiae displayed twohaplotypes that were connected to a central unresolvedloop with P. abencerragus. Eight and nine mutational stepsjoined P. barbagiae with P. abencerragus from North Af-rica and Europe respectively, while thirteen mutationalsteps joined P. barbagiae with P. abencerragus from Israel.In contrast, 16 mutational steps joined P. barbagiae withthe closest haplotype of the A1-A3 haplogroup (includingP. panoptes, P. baton and P. vicrama), a result notreflected in the molecular analyses using both mt and ncmarkers, where P. barbagiae was recovered as sister to theclade including P. panoptes, P. baton and P. vicrama(Fig. 1a, Additional file 3).

Tests of demographic equilibriumAccording to both the Fs and R2 parameters, calculatedfor six mtDNA sequence sets (Table 1), the null hypoth-esis of constant population size could be rejected fortwo phylogeographic units (set in bold in Table 1): P.abencerragus (number of mtDNA sequences (N) = 23)and haplogroup A2 (N = 40) including P. vicrama se-quences and P. baton from Corsica and southern Italy(Fig. 1b). The mismatched distribution of these sequencesets was also examined according to a sudden expansionmodel. Goodness of fit tests indicated significant devia-tions from expected distributions only for P. abencerra-gus that showed a multimodal and ragged shape,revealing demographic equilibrium or a stable popula-tion [53]. Conversely, for the haplogroup (A2) theparameter τ = 2 μ t could be used to estimate the time(t) elapsed from population expansion. According toBrower’s mutation rate [40], the demographic expansionof the haplogroup A2 could be traced back to about 41thousand years ago (kya) (0−207 kya).

Biogeographical analysesThe results of the ancestral area reconstructions in Bio-GeoBEARS are summarized in Tables 2 and 3. Allmodels, which implemented founder-event speciationparameter (+j), were significantly better supported thantheir more simple counterparts. The AIC weights furthershowed the DEC + j model to achieve the highest relativeprobability (70%; see Table 2). However, the geographicalorigin of Pseudophilotes remained unclear, as the DEC +j model indicated several potential area combinations,none of these significant (Fig. 2b). Also the ancestralarea for the P. abencerragus group (Pseudophilotes s.str.) remained unclear, with Eurasia+Iberia+Africahaving the highest probability (60%; see Table 3).More details on the biogeographic modelling aregiven in the discussion.

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DiscussionThe taxonomical status of the genus Pseudophilotes andits speciesMitochondrial and nuclear phylogenetic analyses(Figs. 1b, 2a, Additional file 3) confirmed the findings ofUgelvig et al. [1] that Pseudophilotes and Scolitantidesare closely related, but clearly separated from each other.It therefore seems justified to maintain Pseudophilotesand Scolitantides as distinct genera (e.g. [56]), despitethe fact that other studies have joined them [2, 3]. Ouranalyses further corroborate the dichotomy between theclade of the species P. bavius and P. fatma, (equivalentto what has been proposed as the genus RubrapterusKorshunov, 1987), and Pseudophilotes s. str. This evolu-tionary split between the Rubrapterus clade and theremaining Pseudophilotes follows larval food plant prefer-ences. P. bavius and fatma feed on Salvia spp. [57, 58],whereas Lamiaceae genera other than Salvia (mostly Thy-mus spp., but also Cleonia, Calamintha, and rarely Cus-cuta as a parasite on thymes) are preferred by all otherspecies [3, 59]. Within P. bavius, several subspecies havebeen described in Europe (e.g. hungarica Diószeghy, 1913;macedonicus Schulte, 1958; casimiri Hemming, 1932; seefor example Tshikolovets [3]), but our dataset did not re-veal any clear spatial genetic differentiation below the spe-cies level. P. fatma has sometimes been treated as asubspecies of P. bavius [60, 61], but given morphological

[62] and genetic differences it is probably best regarded asa distinct species.Among the Thymus-feeders, the southern-mediterranean

species P. abencerragus is clearly distinct from all otherspecies. Within P. abencerragus, the sequences obtainedfrom North African and Israeli specimens appear to bederived from those of Iberian individuals, reflecting pre-viously proposed subspecies (North Africa: colonariumTurati, 1927; Israel: nabateus Graves, 1925 and ameliaHemming, 1927). However the support of these rela-tionships is weak, prohibiting final statements on theirtaxonomic status. The Sardinian endemic P. barbagiaebelongs to the baton-group but is clearly separatedfrom the clade comprising P. baton, P. panoptes and P.vicrama. Although species delimitation analyses supportthe status of all of the latter (Fig. 2a), our tree reconstruc-tion analyses (Additional file 3) and the mtDNA haplotypenetwork (Fig. 1b) indicate a more complex patternrepresenting a challenging taxonomic case that requiresfurther study to clarify species boundaries and theirprecise geographic distribution, as well as possiblehybrid zones.

Biogeographical history of the genus PseudophilotesOur results are consistent with other studies on Polyom-matinae [63] indicating that the most recent commonancestor of the genus Pseudophilotes probably lived inthe Messinian period (5.33–7.25 Mya). This ancestorhad already colonized Lamiaceae as larval hosts, a syna-pomorphic character differentiating Pseudophilotes fromScolitantides and Philotes; affiliation with Lamiaceae is arather rare trait in the Polyommatinae worldwide [64, 65].During the Messinian, the closure of the Strait of

Gibraltar led to an almost complete desiccation of theMediterranean Sea, permitting massive faunal exchange be-tween southern Europe and the Maghreb region [23, 24].Within Pseudophilotes s. str., the ancestor of P. abencerra-gus split into the extant P. abencerragus that occurs insouthwestern Europe and from northwestern Africa east-wards through to southern Israel, and the ancestor of P.

Table 2 Results of the BioGeoBEARS model comparisons

LnL dF d e j AIC AIC_wt

DEC − 25.29 2 0.10 0.11 0.00 54.58 0.06

DEC + J −21.80 3 0.04 0.00 0.41 49.60 0.70

DIVALIKE −26.37 2 0.12 0.08 0.00 56.74 0.02

DIVALIKE+J −23.41 3 0.04 0.00 0.38 52.82 0.14

BAYAREALIKE −28.95 2 0.24 0.49 0.00 61.90 0.00

BAYAREALIKE+J −24.01 3 0.03 0.00 0.54 54.03 0.08

Parameters: LnL log-likelihood of the model, d dispersal, e extinction, jfounder-event, AIC Akaike information criterion, ΔAIC difference between thecurrent AIC score and the model with the best AIC score (bold), AICw Akaikeweights: probability of each model being the true model, best model in bold

Table 3 Results of the BioGeoBEARS ancestral area reconstructions with relative probabilities of the reconstructed area(s)

Areas

A B C D E AB AC AD BC BD ABC ABD ACD BCD

Clades

Pseudophilotes 0.17 0.07 0.16 0.01 0.00 0.10 0.14 0.01 0.09 0.01 0.21 0.01 0.01 0.01

Rubrapterus 0.44 0.00 0.44 0.00 0.00 0.00 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Pseudophilotes s. str. 0.02 0.02 0.01 0.01 0.00 0.11 0.06 0.00 0.06 0.01 0.60 0.05 0.03 0.02

P. baton group + P. barbagie 0.19 0.27 0.00 0.31 0.00 0.17 0.00 0.01 0.00 0.01 0.00 0.04 0.00 0.00

P. baton group 0.11 0.25 0.00 0.00 0.00 0.63 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

P. baton + P. vicrama 0.15 0.05 0.00 0.00 0.00 0.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Area codes: Eurasia (A), Iberian Peninsula (B), North Africa (C), Sardinia (D), and North America (E)

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baton, P. panoptes, P. vicrama and P. barbagiae. Althoughclimate modelling analyses [66] showed differences in cli-mate requirements that possibly suggest different evolu-tionary histories for the more western P. baton and itseastern vicariant P. vicrama, the two species are parapatricin Central Europe (Fig. 1a) and all P. baton analysed frompopulations in Corsica share their mtDNA haplotype (H32)with P. vicrama. The large geographical distance of theCorsican populations from the extant contact zone ineastern central Europe prompts some degree of historicalintrogression between these two species. This hypothesis issupported both from the distribution of the H32 haplotype(Fig. 1a; Additional file 1) and the genetic traces of demo-graphic expansion in P. vicrama (haplogroup A2: Fig. 1b)pointing to the Late Pleistocene (about 41 kya), whensea-level oscillations created land bridges between Corsicaand mainland [25] permitting faunal exchange.

Isolation of the Sardinian P. barbagiaeThe Sardinian Blue butterfly, Pseudophilotes barbagiae[6] is restricted to mountainous habitats on the slopes ofthe Gennargentu massif in Central Sardinia. Its larvaedevelop exclusively on Thymus herba-barona, whichmostly grows between 800 and 2000 m [67–69]. InSardinia, this plant is restricted to a few slopes in theBarbagiae and Gennargentu Mountains, and on MountLimbara, although in other Mediterranean regions it hasa much wider distribution. Interestingly, the plant alsooccurs in Corsica, while the butterfly does not. In theInternational Union for Conservation of Nature (IUCN)Red List of Threatened Species [70], P. barbagiae isassessed as Data Deficient, as little is known about thisbutterfly. The thyme species it feeds on has not yet beenassessed by the IUCN, however according to the Euro-pean Red List of Vascular Plants [71] this plant speciesis not threatened. Our analyses of combined mt and ncmarkers show that P. barbagiae appears more closelyrelated to the clade including P. baton, P. vicrama andP. panoptes than to P. abencerragus. On the other hand,the haplotype network (Fig. 1b) depicts a somewhat dif-ferent picture. Here, P. barbagiae results more closelyrelated to P. abencerragus than to P. baton. Suchdiscrepancies between analyses based solely on the COIregion and others based on combined data, includingnuclear markers, are quite common, since the barcodingregion by itself is not sufficient for drawing phylogeneticconclusions and mitochondrial inheritance often followsdifferent pathways that do not necessarily reflect theevolutionary history of species.Our hypothesis is that during the Pleistocene, when

Sardinia was connected with Corsica and the mainlanddue to glacial/interglacial sea-level oscillations [25], geneflow occurred among baton-group European populationsand Sardinia. Afterwards, gene flow among these became

interrupted and from this founder population P. barba-giae evolved into an endemic species about 0.74 Mya.Interestingly, the mtDNA of P. baton from Corsica is ofthe vicrama-type (H32) shared with individuals of P.vicrama distributed from Finland to Iran (Fig. 1b;Additional file 1). An expansion from Africa, as it hasbeen hypothesized for two other Sardinian endemicbutterflies (Maniola nurag: Kreuzinger et al. [72] andPapilio hospiton: Pittaway et al. [73]), does not seema plausible scenario, despite the smaller geographicaldistance between Sardinia and the African continent.

ConclusionsThis study confirmed the monophyly of the genus Pseu-dophilotes and shed further light on the validity of itscomponent species, as described by current taxonomy.We found evidence for a deeper split between the Salviafeeders (P. bavius and P. fatma) and the Thymus feeders(Pseudophilotes s. str.). However, the precise speciesboundaries and possible signals of introgression andhybridization within the baton-group further meritcloser scrutiny. Finally, P. barbagiae turned out to be ayoung endemic, but clearly with European instead ofNorth African origin.

Additional files

Additional file 1: Dataset analysed in this study. Taxon = Pseudophilotesand outgroup species analysed in this study. Sample numbers (N),collection locality, population code, mtDNA haplotype code andGenBank code. (XLSX 24 kb)

Additional file 2: Primer names, references, primer sequences as well asrespective annealing temperatures used to amplify nuclear andmitochondrial markers in the present study. (XLSX 16 kb)

Additional file 3: Maximum-likelihood tree. Maximum-likelihoodphylogeny inferred from the combined genes dataset of Pseudophilotesand outgroups, with bootstrap support (%) indicated for the sevenspecies analysed. (PDF 515 kb)

Abbreviations28S: 28S ribosomal gene; BPP v4.0: Bayesian Phylogenetics andPhylogeography software; BS: Bootstrap support; CCDB: Canadian Centre forDNA Barcoding; COI: Cytochrome c oxidase subunit 1 gene; EF1α: ElongationFactor 1α gene; h: Haplotype diversity; H: Number of unique mtDNAhaplotypes; HPD: Highest probability density; ITS2: Internal TranscribedSpacer 2 gene; IUCN: International Union for Conservation of Nature;kya: Thousand years ago; MCC: Maximum clade credibility tree; MJ: MedianJoining; ML: Maximum Likelihood; MSC: Messinian Salinity Crisis;mt: Mitochondrial; Mya: Million years ago; N: Number of mtDNA sequences;nc: Nuclear; PCR: Polymerase Chain Reaction; pP: Posterior probabilities;RSS: Randomly similar sections; SD: Standard Deviation; wg: Wingless gene

AcknowledgementsWe are extremely grateful to V. Nazari, P. Huemer, A. Hausmann, M. Mutanen,A. Sourakov and B. Dardenne (DNA barcoding Lepidoptera of Upper-Normandyregion’ project) for providing sequences and two anonymous reviewers for theirhelpful comments. Finally, Canadian Centre for DNA Biotecnology (CCDB) isacknowledged for technical support.

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FundingFWF grant V168-B17 to A. Grill, a Marie Curie IOF within the 7th EuropeanCommunity Framework Programme to V. Dincă (project no. 625997), theEuropean Union’s Seventh Framework programme for research and innovationunder the Marie Skłodowska-Curie grant agreement No 609402–2020 researchers:Train to Move (T2M) to R. Vodă, the Russian Science Foundation to the ZoologicalInstitute of the Russian Academy of Sciences (N 14–14-00541) to V. Lukhtanov.Genetic analyses were further supported through funds of the Faculty of LifeSciences, University of Vienna, Austria.

Availability of data and materialsThe datasets generated during the current study are available in the BOLDsystem repository: Dataset name: DS-PSEUDOPH; DOI: dx.doi.org/10.5883/DS-PSEUDOPH.

Specimens collection statementWe declare that all samples analysed in this study were collected complyingwith institutional, national, and international guidelines.

Authors’ contributionsVT, AG and KF designed the study. AG, RV, VD and VL collected samples. VT,BG, RV, VD and VL sequenced the specimens. VT, HL and VD analysed thedata. VT, AG, HL and KF wrote the manuscript. All authors edited themanuscript and approved its final version.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Botany and Biodiversity Research, Division of TropicalEcology and Animal Biodiversity, University of Vienna, Rennweg 14, A-1030Vienna, Austria. 2Department of Ecology and Genetics, University of Oulu, POBox 3000, 90014 Oulu, Finland. 3Institut de Biologia Evolutiva(CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta 37, 08003Barcelona, Spain. 4DBIOS, Dipartimento di Scienze della Vita e Biologia deiSistemi, Università degli Studi di Torino, Via Accademia Albertina 13, 10123Turin, Italy. 5Department of Karyosystematics, Zoological Institute of RussianAcademy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia.6Department of Entomology, St. Petersburg State University, Universitetskayanab. 7/9, St. Petersburg 199034, Russia.

Received: 15 November 2017 Accepted: 19 June 2018

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